Augmentation or replacement of the bladder is often necessary for the treatment of adults with bladder cancer and children with spinal cord injury or spina bifida. Current surgical techniques utilize segments of intestine or stomach as a substitute for bladder wall. Use of intestinal segments is associated with many complications including infection, stones, salt imbalance, and most concerning, cancer. An ideal substitute for bladder wall would be bioengineered bladder tissue. Ideally, a bioengineered graft would consist of cells that are genetically normal and free of cancerous mutations, promote blood vessel growth, survive long-term and regenerate. Stem cells appear to be the ideal solution for bioengineering tissue.
Preliminary clinical trials have demonstrated the feasibility of using bioengineered tissue for bladder augmentation. The bladder is lined by a very unique cell type called “urothelium”. The ability to induce human embryonic stem cells (hESC) or induced pluripotent stem cells (iPSC) into urothelium would provide a major advancement in the tissue engineering field, scientifically and clinically. In addition, deciphering the mechanisms of hESC to urothelial differentiation would facilitate investigation of deviated differentiation into urothelial cancer stem cells; the “seeds” of bladder cancer.
Bladder cancer is the fourth most common type of cancer and caused 15,000 deaths last year. Treatment often requires removal of the bladder. Like other tumors, bladder cancer is believed to originate from the transformation of stem cells into cancer stem cells (CSCs). Potential markers of urothelial CSCs have been identified. Surprisingly, the scientific community has not yet addressed the study of normal human urothelial stem cells and differentiation of hESC to urothelium. The investigation of mechanisms and markers involved in the differentiation of hESC into urothelium will yield important facts about normal and abnormal differentiation and will ultimatley help predict the malignancy of bladder cancers and improve treatments.
Our specific aims are to induce the differentiation of hESC into urothelium via cell signaling. We will also investigate the genes involved in this process. And, we will test the feasibility of transplanting hESC-derived urothelium into a bladder.
This investigation will lead to advances in stem cell biology in an important area not addressed by other scientists. The successful completion of this project will improve human health, indirectly through increased knowledge of differentiation pathways relevant to normal development and neoplasia, and directly through development of novel methodologies for bioengineering tissue for adults and children with urologic disorders and cancer. We are working in a very novel field, which has a high potential to save lives and to vastly improve the quality of life for many patients who need their bladder removed or enlarged.

Statement of Benefit to California:

The scientific community has not yet addressed the study of urothelial stem cells and differentiation of human embryonic stem cells (hESC) to urothelium. Our investigation of mechanisms and markers involved in the differentiation of hESC into urothelium will yield important facts about normal and abnormal differentiation and will help predict the malignancy of bladder cancers and improve treatments. This project will also advance the field of regenerative medicine. Adults with bladder cancer and children with spina bifida often need bladder reconstruction. Current surgical techniques use segments of intestine as a substitute for bladder wall. Use of intestinal segments is associated with many complications including cancer. Preliminary clinical trials have demonstrated the feasibility of using bioengineered tissue. The ability to induce hESC or induced pluripotent stem cells (iPSC) into urothelium would provide a major advancement in the regenerative medicine field, both scientifically and clinically.
Due to its high rate of recurrence, bladder cancer carries the highest lifetime cost to treat of all cancers. The successful completion of this project will improve human health, indirectly through increased knowledge of differentiation pathways relevant to normal bladder development and bladder cancer, and directly through development of novel methodologies for bioengineering tissue for adults and children with urologic disorders and cancer. These benefits will come to the citizens of California first. In addition to healthcare, this research will benefit the California economy by developing new protocols and technologies that could be adapted for other organs and tissues. Any health benefits, patents, new biotechnology or clinical trials would start in California. This research exemplifies the intent of CIRM bringing together clinical scientists with basic and translational scientists to develop stem cell treatments for the California public while at the same time advancing stem cell biology.

Progress Report:

Year 1

Augmentation or replacement of the bladder is often necessary for the treatment of adults with bladder cancer and children with spinal cord injury or spina bifida. Current surgical techniques utilize segments of intestine or stomach as a substitute for bladder wall. Use of intestinal segments is associated with many complications including infection, stones, salt imbalance, and most concerning, cancer. An ideal substitute for bladder wall would be bioengineered bladder tissue. Stem cells appear to be the ideal solution for bioengineering tissue. The ability to induce human embryonic stem cells (hESC) or induced pluripotent stem cells (iPSC) into urothelium, the cells that line the bladder, would provide a major advancement in the tissue engineering field, scientifically and clinically.
Our specific aims are to induce the differentiation of hESC into urothelium via cell signaling. We will also investigate the genes involved in this process. And, we will test the feasibility of transplanting hESC-derived urothelium into a bladder.
In the last year we have made progress developing a technique for the induction of human embryonic stem cells into urothelium. In the developing embryo, embryonic stem cells are preprogrammed to turn into urothelium when induced by the appropriate and timely signals. The challenge is to simulate this in a culture dish. We have found that embryonic stem cells can be induced to form urothelium when they are exposed to growth factors released by cultured adult urothelium. We have done this by growing them together (without contact) and currently are testing solutions with these growth factors. We are also studying the molecular mechanisms and genes involved in this process.
We have also made significant progress on our third aim which is to test the feasibility of transplanting cultured urothelium. In a small animal model we have been able to reliably transplant these cells into the bladder and demonstrate their survival and incorporation into the bladder. We are currently testing better techniques of transplantation and following the animals longer to determine the life span of these cells.
This investigation will lead to advances in stem cell biology in an important area not addressed by other scientists. The successful completion of this project will improve human health, indirectly through increased knowledge of differentiation pathways relevant to normal development and neoplasia, and directly through development of novel methodologies for bioengineering tissue for adults and children with urologic disorders and cancer. We are working in a very novel field, which has a high potential to save lives and to vastly improve the quality of life for many patients who need their bladder removed or enlarged.

Year 2

The first aim of this project was to induce human embryonic stem cells (hESCs) into urothelium, which is the epithelial lining of the urinary tract. These cells are very specialized and unique. They are coated with a group of proteins, named uroplakins, which make the cells uniquely impermeable to many contaminants in the urine. Uroplakins are almost exclusively produced by urothelial cells, so they serve as a unique marker for these cells, and hence high expression of uroplakin is considered sine qua non for identifying urothelium. Thus, we are able to identify urothelial cells based on their expression of uroplakin proteins on the cell surface, as well as inside the cells, using various experimental techniques.
We evaluated numerous conditions to induce hESCs into urothelium. The first step in doing this was to induce hESCs into definitive endoderm (DE), which is a precursor stage of epithelial differentiation for the cells lining the intestines and urinary tract. We did this very efficiently and found that specialized urothelial cell culture medium with growth factor additives (uromedia) that we have optimized in our laboratory was able to induce about 20-30% of DE cells into urothelium, as determined by uroplakin expression. Since urothelium makes up less than 1% of the cells in the body, we believe we have developed a system that induces hESCs beyond their spontaneous preprogramming. We also tested the ability of growing DE with adult urothelium or adding growth factors produced by cultured adult urothelium or bladder smooth muscle to augment the differentiation, none of which succeeded in increasing urothelial cell differentiation in a statistically significant manner.
We were able to isolate the hESC-derived urothelium using a technique that sorts live cells based on protein expression (Fluorescence Activated Cell Sorting, or FACS). These cells, sorted based on uroplakin expression on the cell surface, are currently being cultured and tested to determine their protein expression and function.
The second aim of the project was to find transcription factors (TF) that are involved in directing the differentiation of hESCs into urothelium. Transcription factors affect the genetic mechanisms that lead to protein production. In our case, we were interested in what TFs were involved in the early expression of uroplakins as DE became urothelium. As there are hundreds of known TFs, we focused on those that have potential binding sites near the uroplakin genes. Using various techniques, we found that certain TFs were expressed in cells at the same time as uroplakins were first appearing. We were also able to see certain TFs disappear as hESCs became DE and then urothelium. Further work needs to be done to determine if these TFs are actually controlling the expression of the uroplakin genes or whether their co-expression is coincidental.
The third aim of the project was to determine if urothelium could be transplanted into a rodent bladder, survive and possibly incorporate into the organ. For injection, we used cultured urothelial cells that were labeled with a fluorescent genetic marker (green fluorescence protein – GFP) that allows them to be distinguished from the colorless host cells. GFP urothelial cells were obtained either from transgenic rats (all the cells in their body express GFP) or from urothelium infected with a specialized virus that transfers the GFP gene into the cells. GFP-urothelium was injected into the bladder walls of mice and rats. Ideally these cells should be injected under the bladder lining of these bladders. This potential space is microscopic and is difficult to infiltrate even with bladders outside the body on the laboratory bench. Therefore, getting into this space in a live animal under anesthesia was very difficult. Primarily, the cells were injected into the lamina propria, which is a space between the bladder muscle and epithelium. We were able to find viable transplanted cells within a few days of injection, but could not find cells at late time points after transplantation. This could be due to the cells not living, or it could be that very few cells did survive, but we were unable to find them.
Future work: We will continue to evaluate the urothelium produced from hESCs in this project. We hope that we can show that these cells appear and function just like normal urothelium, and thus be used for future tissue engineering applications.